ABSTRACT
MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. Here, we performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.
Subject(s)
Drosophila Proteins , MicroRNAs , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Drosophila/genetics , Drosophila/metabolism , RNA, Messenger/genetics , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Mammals/geneticsABSTRACT
New derivatives of phaeosphaeride A (PPA) were synthesized and characterized. Anti-tumor studies were carried out on the U937, HCT-116, PC3, MCF-7, A549, Ð562, NCI-H929, Jurkat, THP-1, RPMI8228 tumor cell lines, and on the HEF cell line. All the compounds synthesized were found to have better efficacy than PPA towards the tumor cell lines mentioned. Compound 6 (IC50â¯=â¯0.59⯱â¯0.27⯵M) was observed to be 11 times more active than PPA (IC50â¯=â¯6.5⯱â¯0.30⯵M) towards the NCI-H929 cell line, with a therapeutic index of 18. Compound 6 was determined to be over half and 16 times more active than etoposide towards the NCI-H929 (IC50â¯=â¯0.9⯱â¯0.05⯵M) and A549 (IC50â¯=â¯100⯱â¯7.0⯵M) cell lines, respectively.
Subject(s)
Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
New derivatives of phaeosphaeride A (PPA) were synthesized and characterized. Anti-tumor activity studies were carried out on the HCT-116, PC3, MCF-7, A549, Ð562, NCI-Ð929, Jurkat, THP-1, RPMI8228 tumor cell lines, and on the HEF cell line. All of the compounds synthesized were found to have better efficacy than PPA towards the tumor cell lines mentioned. Compound 6 was potent against six cancer cell lines, HCT-116, PC-3, K562, NCI-H929, Jurkat, and RPMI8226, showing a 47, 13.5, 16, 4, 1.5, and 7-fold increase in anticancer activity comparative to those of etoposide, respectively. Compound 1 possessed selectivity toward the NCI-H929 cell line (IC50 = 1.35 ± 0.69 µM), while product 7 was selective against three cancer cell lines, HCT-116, MCF-7, and NCI-H929, each having IC50 values of 1.65 µM, 1.80 µM and 2.00 µM, respectively.
